Intracellular and Mitochondrial Reactive Oxygen Species Measurement in Primary Cultured Neurons
原代培养的神经元细胞和线粒体中活性氧的测定   

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The Journal of Neuroscience
May 2017

 

Abstract

Reactive oxygen species (ROS) are chemically reactive oxygen containing molecules. ROS consist of radical oxygen species including superoxide anion (O2•−) and hydroxyl radical (•OH) and non-radical oxygen species such as hydrogen peroxide (H2O2), singlet oxygen (O2). ROS are generated by mitochondrial oxidative phosphorylation, environmental stresses including UV or heat exposure, and cellular responses to xenobiotics (Ray et al., 2012). Excessive ROS production over cellular antioxidant capacity induces oxidative stress which results in harmful effects such as cell and tissue damage. Sufficient evidence suggests that oxidative stresses are involved in cancers, cardiovascular disease, and neurodegenerative diseases including Alzheimer’s disease and Parkinson disease (Waris and Ahsan, 2006). Though excessive level of ROS triggers detrimental effects, ROS also have been implicated to regulate cellular processes. Since ROS function is context dependent, measurement of ROS level is important to understand cellular processes (Finkel, 2011). This protocol describes how to detect intracellular and mitochondrial ROS in live cells using popular chemical fluorescent dyes.

Keywords: Reactive oxygen species (ROS) (活性氧族(ROS)), Intracellular ROS (细胞内ROS), MitoSOX (MitoSOX), CM-H2DCFDA (CM-H2DCFDA), Primary neuron (原代神经元)

Background

ROS are important to maintain homeostasis in our bodies (Brieger et al., 2012). Many diseases such as cancer, neurodegenerative disease, cardiovascular disease, and diabetics are associated with ROS (Datta et al., 2000). DNA damage caused by ROS is a major cause of accelerating carcinogenesis process, and therapeutic agents targeting ROS have been actively developed (Trachootham et al., 2009). In circulatory system, abnormal oxidative stress increases the production of ROS, leading to various cardiovascular diseases (Forstermann, 2008). Signaling related to diabetes is sensitive to ROS, and these signaling abnormalities induced by abnormal levels ROS cause diabetes complications (Baek et al., 2017). Controlling the ROS levels in the brain is one of the most important activities because abnormal levels of ROS can cause diverse brain diseases. Amyloid beta, known as an important factor in Alzheimer’s disease, causes excessive ROS generation in the brain, neuronal damage (Singh et al., 2011), and eventually dementia (Polidori, 2004). Activated microglia produced by ROS which secretes a variety of cytokines result in neuronal death (Heneka et al., 2014).

ROS are generated by small part of oxygen consumed in mitochondria. A principal species of ROS produced in mitochondria is superoxide anion and it is the byproduct of the electron transport chain (Batandier et al., 2002). In order to detect superoxide in mitochondria, MitoSOX red, a mitochondria superoxide indicator, is used. Due to the positive charge on triphenylphosphonium group, MitoSOX red can effectively penetrate phospholipid bilayer, and accumulate into the matrix of mitochondria. Furthermore, hydroethidine of MitoSOX red allows researchers to discriminate the fluorescent signal generated by superoxide-mediated oxidative products from other non-specific signals (Robinson et al., 2006; Baek et al., 2017).

CM-H2DCFDA is a chloromethyl derivative of H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate), a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity within the cell and can be used to detect the intracellular formation of ROS (Kirkland et al., 2007). Once the fluorescent probe of CM-H2DCFDA permeates cell membrane, intracellular esterases hydrolyze its acetyl groups and it can be retained in the cell. CM-H2DCFDA is more sensitive to oxidation by H2O2 than superoxide (O2•−) (Fowler et al., 2017). CM-H2DCFDA is widely used in physiological and pathophysiological studies including virus infection (Nykky et al., 2014), cancer (Khatri et al., 2015; Liu et al., 2017), and neurodegenerative diseases (Ng et al., 2014). Using CM-H2DCFDA, we can detect intracellular ROS level by flow cytometry/fluorescence measurement and the localization of ROS producing organelle with confocal microscopy (Forkink et al., 2010).

Materials and Reagents

  1. Glass bottom cell culture dish type 35 mm and dimension 20 mm (Nest Scientific, catalog number: 801001 )
  2. Cover glasses thickness No. 1 circular size 18 mm Ø (MARIENFELD, catalog number: 0111580 )
  3. Petri dish, 100 mm Polysterene aseptic non-tissue culture treated (SPL Life Sciences, catalog number: 10095 )
  4. 15 ml conical tube (SPL Life Sciences, catalog number: 50015 )
  5. 10 ml Serological pipettes (SPL Life Sciences, catalog number: 91010 )
  6. 50 ml conical tube (SPL Life Sciences, catalog number: 50050 )
  7. Cell strainer 70 μm (Corning, Falcon®, catalog number: 352350 )
  8. Pregnant female Sprague Dawley rats (E17-E18 days gestation, Orient Korea)
  9. Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P6407-5mg )
  10. Phosphate buffered saline powder, pH 7.4, for preparing 1 L solutions (Sigma-Aldrich, catalog number: P3813 )
  11. CM-H2DCFDA (Thermo Fisher Scientific, InvitrogenTM, catalog number: C6827 )
  12. Dimethyl Sulfoxide(DMSO) (Merck, catalog number: 317275 )
  13. MitoSOXTM Red Mitochondrial Superoxide Indicator, for live-cell imaging (Thermo Fisher Scientific, InvitrogenTM, catalog number: M36008 )
  14. Phosphate buffered saline (PBS) powder, pH 7.4, for preparing 1 L solutions, suitable for cell culture(Sigma-Aldrich, catalog number: P3813 )
  15. Trypsin (2.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
  16. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  17. Neurobasal Medium® (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
  18. B-27TM Supplement (50x), serum free (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
  19. DMEM High Glucose (4.5 g/L), with L-Glutamine, with Sodium Pyruvate (Capricorn Scientific, catalog number: DMEM-HPA )
  20. Penicillin/Streptomycin (100x) (PS) (Capricorn Scientific, catalog number: PS-B )
  21. Amyloid beta peptide 1-42 Human (ANYGEN, catalog number: AGP-8338 )
  22. CM-H2DCFDA solution (see Recipes)
  23. MitoSOXTM Red solution (see Recipes)
  24. Poly-D-lysine hydrobomide solution (see Recipes)
  25. Prep medium (see Recipes)
  26. Culture medium (see Recipes)
  27. Maintain culture medium (see Recipes)

Equipment

  1. Haemacytometers (MARIENFELD, catalog number: 0630010 )
  2. Original Portable Pipet-Aid® Pipette Controller (Drummond Scientific, catalog number: 4-000-100 )
  3. Dressing Scissors (Surgimax Instruments, catalog number: 85-112-12 ) (Figure 1A 1)
  4. Dissecting Scissors (Surgimax Instruments, catalog number: 85-127-10 ) (Figure 1A 2)
  5. Dissecting Scissors (Surgimax Instruments, catalog number: 63-175-11 ) (Figure 1A 3)
  6. Spring Dressing Forceps Sharp (Surgimax Instruments, catalog number: 85-076-11 ) (Figure 1A 4)
  7. Spring Dressing Forceps Blunt (Surgimax Instruments, catalog number: 85-073-15 ) (Figure 1A 5)
  8. Multi Purpose Forceps Pointed (Surgimax Instruments, catalog number: 05-177-11 ) (Figure 1A 6)
  9. Clean bench (HANBAEK Scientific Technology, catalog number: HB-402 )
  10. Cell culture CO2 incubator (ARA, catalog number: APR150 )
  11. Water-bath (Grant Instruments, JB Academy, catalog number: JBA18 )
  12. Centrifuge (Hanil Scientific, catalog number: Combi 514R )
  13. Confocal microscope with live cell imaging system (Carl Zeiss, model: LSM700 ) (Figures 1B and 1C)


    Figure 1. Equipment for the experiment. A. Surgery instruments; B. Confocal microscope (LSM700) with live cell imaging system; C. Live cell chamber.

Software

  1. For measure
    1. ZEN black version (ZEISS confocal microscope LSM700 software)
      Note: This is default program provided with ZEISS confocal microscope.

  2. For analysis
    1. ZEN Blue edition (ZEISS confocal microscope LSM700 software; SR-DIP software for ZEN blue edition)
    2. ImageJ (ImageJ is an open source image processing program)

Procedure

In this protocol, we introduce two types of ROS measurement in primary neuronal cells under oxidative stress.

  1. In advance, a 35 mm plate for live cell imaging, is coated with 2 ml poly-D-lysine solution at 4 °C for 24 h.
    Optional: Instead of using 35 mm dish for ROS measurement, the following method is possible. Place the sterile cover glasses slip flat into each well of a 6-well plate. Add 2 ml of Poly-D-lysine solution to each well and coat at 4 °C for 24 h.
  2. Remove the poly-D-lysine solution and wash twice with cold PBS.
  3. Dry the coated plate on a bench while E17 rat embryo is being prepared.
  4. Carefully take out the E17~E18 embryos from the Sprague Dawley rat using Dressing Scissors (Equipment 3) and Spring Dressing Forceps Blunt (Equipment 7). Place them in a Petri dish filled with cold Prep medium (Figures 2A-2D).
    Note: In this experiment, the pregnant Sprague Dawley rats were anesthetized with CO2 and euthanized using CO2 after embryos extraction. All experimental procedures were conducted after approval of Institutional Animal Care and Use Committee of Sungkyunkwan University.
  5. Using Dissecting Scissors (Equipment 5) and Spring Dressing Forceps Sharp (Equipment 6), carefully take out individual embryo from uterus and embryonic sack (Figures 2E-2F).
  6. Using Dissecting Scissors (Equipment 4) and Multi Purpose Forceps Pointed (Equipment 8), excise the scalp and skull of the E17-E18 embryos and pull out the whole brain. Place the extracted brain in Prep medium (Figures 2G-2I).
    Note: For proper cell conditions, this process (Steps 4-6) should be finished within 15-20 min.


    Figure 2. The process of embryo extraction in pregnant Sprague Dawley rat (E17-E18) (Steps 4-6)

  7. After taking out the embryo's brain, place it in the prep medium as shown in Figure 3A.
  8. Dissect the cerebral cortex from the whole brain as Figure 3B.
    Note: When separating the cortex from the embryo’s brain, you must be careful not to separate other parts together. When you progress from Step 7 to Step 8 (Figure 3A to Figure 3B), insert the micro forceps between the inner side of the cortex and the outer part of the striatum, and then cut cortex from embryo’s brain. As you see in Figure 3A, the cortex is on the surface as it envelops other parts of the brain. There is a striatum on the inner side just below the cortex.
  9. Remove meninges and blood vessels outside the cerebral cortex (Figure 3C). Place the cerebral cortex in Prep medium as Figure 3D.
    Notes:
    1. After removing the meninges and blood vessels as shown in Figure 3, remove the hippocampus part that is attached to the inner side of the cortex. You can use the cortex part for cortical neuron cell culture or you can conduct hippocampal neuron cell culture with the hippocampus part.
    2. For proper cell conditions, this process (Steps 7-9) should be done within 20-25 min.


    Figure 3. The process of extracting cortex region from the extracted whole brain (Steps 7-9)

  10. Transfer the cerebral cortex to a conical tube (15 ml size) filled with 2 ml of Prep medium and add 300 μl of trypsin solution to make final concentration about 10% (Figure 4A).
  11. Place the conical tube in a 37 °C incubator or water bath and gently tapping it periodically (Figure 4B).
  12. After about 15 min, carefully suspend the cells using a 10 ml disposable pipette (Figure 4C).
  13. Put 400 μl of FBS in the tubes to 10% concentration and carefully suspend the cells again.
  14. Filter the cells through a 70 μm nylon cell strainer to obtain single cell suspension (Figure 4D).
  15. Seed the cells at 1 x 106 cells in a 35 mm dish after diluting the cells with 2 ml Culture medium and incubate the cells in an incubator at 37 °C for 18 h (Figures 4E and 4F).
  16. Replace Culture medium in the dish with 2 ml of Maintain Culture medium.
    Note: If cells are maintained in the Culture medium for too long, other brain cell types including microglia and astrocyte are likely to grow up. Therefore, Culture medium should be replaced with Maintain culture medium 12-18 h after the cell seeding.
  17. Replace the half of the media in the 35 mm dish with the new 1 ml Maintain culture medium every other day.


    Figure 4. The Process of making single cells of brain tissue and preparing for cell seeding (Steps 10-17)

  18. Grow the cells for at least 7 days after the seeding for experiments (Figure 5).
    Note: When you seed the cells, various brain cells (neuron, microglia, astrocytes) are present in the medium. However, after replacement with the Maintain culture medium, only the neuron cells remain specifically because all other types of cells except for the neuron cell are removed by the B-27 constituting the Maintain culture medium.


    Figure 5. Cell morphology at the 1st day (A) and after 8 days (B). Scale bars, A = 500 μm; B = 200 μm.

  19. Incubate the cells in ROS inducing conditions for about 24 h.
    Note: 5 μM of oligomeric Aβ1-42 was added to induce ROS in this protocol, but it may vary depending on your experimental conditions. Aβ1-42 is already known to induce ROS (Andrey et al., 2004, Shelat et al., 2008).
  20. And then treat with 1 μl of CM-H2DCFDA solution (5 mM) or MitoSOXTM Red solution (5 mM) for 20 min.
    Note: MitoSOXTM is a specific indicator for mitochondrial superoxide and CM-H2DCFDA is more sensitive to oxidation by H2O2 than superoxide (O2•−). Therefore, even if two chemicals are processed at the same time, they are labeled with different ROS. However, it is difficult to distinguish exactly two types of ROS and analyze fluorescence in the confocal image. We recommend preparing samples separately and conduct each experiment.
    Optional: This section provides another option for preparing samples to capture live cell images. If you have cultured cells on a coverslip, carefully put the coverslip into a new 35 mm live cell imaging dish. Put the side of coverslip that cells are attached to the bottom surface.
  21. Replace with the new 2 ml of Maintain culture medium.
    Note: Pre-set the condition for taking live cell imaging; 37 °C and 5% CO2 is required because we want to keep the growing conditions for primary neuron cells.
  22. Using confocal microscope, measure the fluorescence mediated by MitoSOXTM (510/580 nm, see Figure 6) or CM-H2DCFDA (495/520 nm, see Figure 7). Since the fluorescence will not last long, it is recommended to measure within 30 min.


    Figure 6. Detection of superoxide in rat primary neuronal cells’ mitochondria with MitoSOXTM Red. Oxidation of MitoSOXTM Red reagent by superoxide produces red fluorescence. A. Bright field rat primary cell images; B. Red fluorescence generated by superoxide; C. Merged image. Scale bars = 50 μm.


    Figure 7. Measurement of ROS in intracellular compartment induced by Aβ in rat primary cells using CM-H2DCFDA. Green fluorescence represents intracellular ROS level of Aβ or vehicle treated sample. A. Vehicle-treated sample; B. 5 μM of Aβ treated sample. Scale bars = 50 μm.

    Confocal image measurement setting
    You can follow the processes in Figure 8 to measure the level of ROS using confocal microscope:
    1. Run the Zen black edition.
    2. Click the ‘Smart Setup’ button in ‘Acquisition’ tab to choose the appropriate excitation/emission wavelengths of the dye (MitoSOXTM or CM-H2DCFDA) (Figure 8 A1).
    3. Set the appropriate excitation/emission wavelengths of the dye and choose the image colors in the ‘Search’ tab (Figure 8C 2).
    4. Click the ‘Best signal’ button.
    5. Put the sample on the live cell imaging chamber (Figure 1C).
    6. Click the ‘Locate’ tab and adjust the focus of confocal microscope (Figure 8B 3).
    7. After setting the focus of the microscope, click the ‘Set Exposure’ button in ‘Acquisition’ tab.
      Note: ‘Set Exposure’ automatically adjusts the detector Gain value (Figure 8A).
    8. Click the ‘Live’ button (Figure 8A).
      Note: ‘Live’ performs constant scanning of real-time image.
    9. To get clearer and more accurate images in 'Live' conditions, adjust each value of ‘Gain’, ‘Digital offset’, ‘Digital Gain’, ‘Pinhole’, and laser power (Figure 8D 5). You can also adjust ‘Speed’ and ‘Averaging’ to acquire better images.
      Note: In this experiment, ‘Speed’ was set to 7 and ‘Averaging’ number was set to 8. (Figure 9)
    10. Click the ‘Snap’ button to acquire the image (Figure 8A).
      Note: All samples should be measured under the same conditions. The measurement method was based on the instructions of the confocal equipment.


    Figure 8. Flow chart of measuring method of ZEN black version of image measurement program


    Figure 9. The tab to control 'speed' and 'averaging' in ZEN black version

Data analysis

The confocal image that measure the ROS can be used for statistical analysis by quantifying the intensity of fluorescence. This protocol offers two methods.

  1. Measurement method using basic confocal drive program (see Figure 10).


    Figure 10. Analysis flowchart of ZEN blue version. Run Zen blue version, an analysis tool provided by Zeiss confocal equipment. Open the image you want to analyze and click the ‘Analysis’ tab. Press the ‘Setup Image Analysis’ button to set the analysis method (see Figure 10-1). Enter the proper analytical condition in order. In particular, set the appropriate threshold value (When you click on the area where CM-H2DCFDA emits fluorescence, the default value is automatically set) in the 3rd step (The analysis setting value must be set up based on the positive control. The settings of all images to be analyzed should be applied equally). Identify the area you want to measure as shown in Figure 10-4. Set the results (Fluorescence mean, Standard deviation, Fluorescence dot number, etc.) you want to obtain and press the ‘Finish’ button. Finally, check the value by pressing the ‘Analysis’ button.

  2. Measurement method using ImageJ (see Figure 11)


    Figure 11. Analysis flowchart of ImageJ. To measure the intensity of fluorescence using ImageJ, several preliminary steps are required. After opening the image you want to analyze, you have to separate the fluorescent area that you want to measure (In flowchart 3, set the range to be measured while adjusting the threshold value. see Figure 11-2, 11-3). Click on the 'Set Measurements' tab as shown in flowchart 4 to set the results you want to obtain. Click the 'Measure' tab and acquire the results.

Notes

  1. Each experimental group should be treated with MitoSOXTM or CM-H2DCFDA twenty minutes before measuring the fluorescence. You should not treat MitoSOXTM and CM-H2DCFDA in the experimental samples at the same time (Step 19).
  2. If the cell growing conditions are not maintained, value of ROS measurement will be inaccurate. So, at least 10 minutes of stabilization time should be given before taking confocal images (Step 21).
  3. When culturing primary neurons, delaying the medium replacement after cell seeding results in a decrease in the percentage of neurons in the cultured cells (Step 16).

Recipes

  1. CM-H2DCFDA solution (5 mM)
    Dissolve 50 μg CM-H2DCFDA (50 μg/1 vial) in 17 μl DMSO
  2. MitoSOXTM Red solution (5 mM)
    Dissolve 50 μg MitoSOXTM Red (50 μg/1 vial) in 13 μl DMSO
  3. Poly-D-lysine hydrobromide solution
    Poly-D-lysine hydrobromide (5 mg/vial)
    50 ml sterile Ultra pure water
  4. Prep medium
    PBS 45 ml
    5 ml PS
  5. Culture medium
    500 ml DMEM
    50 ml FBS
    5 ml PS
  6. Maintain culture medium
    50 ml Neurobasal media
    1 ml B-27 supplement
    500 μl PS

Acknowledgments

This research was supported by grants (2012R1A5A2A28671860, 2017M3C7A1048268) funded by the Basic Science Research Program through the National Research Foundation of Korea (NRF), the Ministry of Education, Science and Technology, Republic of Korea. The authors state that they have no conflict of interest to declare.

Reference

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  3. Batandier, C., Fontaine, E., Keriel, C. and Leverve, X. M. (2002). Determination of mitochondrial reactive oxygen species: methodological aspects. J Cell Mol Med 6(2): 175-187.
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  6. Finkel, T. (2011). Signal transduction by reactive oxygen species. J Cell Biol 194(1): 7-15.
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  8. Fowler, T. L., Fisher, M. M., Bailey, A. M., Bednarz, B. P. and Kimple, R. J. (2017). Biological characterization of a novel in vitro cell irradiator. PLoS One 12(12): e0189494.
  9. Forkink, M., Smeitink, J. A., Brock, R., Willems, P. H. and Koopman, W. J. (2010). Detection and manipulation of mitochondrial reactive oxygen species in mammalian cells. Biochim Biophys Acta 1797(6-7): 1034-1044.
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简介

活性氧物质(ROS)是化学活性的含氧分子。 ROS由自由基氧物种组成,包括超氧化物阴离子(O2-)和羟基自由基(·OH)以及非自由基氧物种如过氧化氢(H
【背景】ROS对维持我们体内的稳态很重要(Brieger等人,2012年)。许多疾病如癌症,神经退行性疾病,心血管疾病和糖尿病与ROS有关(Datta等人,2000)。由ROS引起的DNA损伤是加速癌变过程的主要原因,并且已经积极开发靶向ROS的治疗剂(Trachootham等人,2009)。在循环系统中,异常的氧化应激增加ROS的产生,导致各种心血管疾病(Forstermann,2008)。与糖尿病有关的信号对ROS敏感,并且由异常水平ROS引起的这些信号异常导致糖尿病并发症(Baek等人,2017)。控制大脑中的ROS水平是最重要的活动之一,因为异常水平的ROS会导致多种脑部疾病。被称为阿尔茨海默病重要因素的淀粉状蛋白β导致脑中过量的ROS生成,神经元损伤(Singh等,2011),最终导致痴呆(Polidori,2004)。由活性氧产生的活性小胶质细胞分泌各种细胞因子导致神经元死亡(Heneka等人,2014)。

ROS是由线粒体中消耗的一小部分氧产生的。线粒体中产生的ROS的主要种类是超氧化物阴离子,它是电子传递链的副产物(Batandier等人,2002)。为了检测线粒体中的超氧化物,使用MitoSOX红色,线粒体超氧化物指示剂。由于三苯基鏻基团带正电,MitoSOX红可以有效地穿透磷脂双分子层,并积聚在线粒体基质中。此外,MitoSOX红的氢化乙啶可使研究人员区分超氧化物介导的氧化产物与其他非特异性信号产生的荧光信号(Robinson等人,2006; Baek等人, 2017)。

CM-H 2 DCFDA是H 2 DCFDA(2',7'-二氯二氢荧光素二乙酸酯)的氯甲基衍生物,其是测量羟基,过氧化氢和其它ROS活性的荧光染料在细胞内并且可以用于检测细胞内ROS的形成(Kirkland et al。,2007)。一旦CM-H 2 DCFDA的荧光探针渗透细胞膜,细胞内酯酶水解其乙酰基团并且其可保留在细胞中。 CM-H 2 DCFDA对H 2 O 2氧化比超氧化物(O 2)更敏感> - )(Fowler et al。,2017)。 CM-H 2 DCFDA广泛用于包括病毒感染(Nykky等人,2014),癌症(Khatri等人, ,2015; Liu等人,2017)和神经变性疾病(Ng et al。,2014)。使用CM-H 2 DCFDA,我们可以通过流式细胞术/荧光测量和利用共焦显微术定位ROS产生细胞器来检测细胞内ROS水平(Forkink等人,2010年)。

关键字:活性氧族(ROS), 细胞内ROS, MitoSOX, CM-H2DCFDA, 原代神经元

材料和试剂


  1. 玻璃底细胞培养皿类型35毫米和尺寸20毫米(巢科学,目录号:801001)
  2. 盖玻片厚度1号圆形大小18毫米Ø(MARIENFELD,目录号:0111580)
  3. 培养皿,100毫米Polysterene无菌非组织培养物处理(SPL Life Sciences,目录号:10095)

  4. 15 ml锥形管(SPL Life Sciences,目录号:50015)
  5. 10毫升血清移液器(SPL Life Sciences,产品目录号:91010)
  6. 50毫升锥形管(SPL Life Sciences,目录号:50050)
  7. 细胞过滤器70微米(Corning,Falcon ,目录号:352350)
  8. 怀孕雌性Sprague Dawley大鼠(E17-E18天妊娠,东方朝鲜)
  9. 聚-D-赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P6407-5mg)
  10. 用于制备1L溶液(Sigma-Aldrich,目录号:P3813)的磷酸盐缓冲盐水粉末,pH7.4。
  11. CM-H 2 DCFDA(Thermo Fisher Scientific,Invitrogen TM,目录号:C6827)。
  12. 二甲基亚砜(DMSO)(Merck,目录号:317275)
  13. 用于活细胞成像的MitoSOX TM线粒体超氧化物指示剂(Thermo Fisher Scientific,Invitrogen TM,目录号:M36008)

  14. 磷酸盐缓冲盐水(PBS)粉末,pH 7.4,用于制备适合细胞培养的1 L溶液(Sigma-Aldrich,目录号:P3813)
  15. 胰蛋白酶(2.5%),无酚红(Thermo Fisher Scientific,Gibco TM,目录号:15090046)
  16. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10082147)
  17. Neurobasal Medium(Thermo Fisher Scientific,Gibco TM,产品目录号:21103049)
  18. B-27 TM补充物(50x),无血清(Thermo Fisher Scientific,Gibco TM,目录号:17504044)
  19. DMEM高葡萄糖(4.5 g / L),含L-谷氨酰胺,丙酮酸钠(Capricorn Scientific,目录号:DMEM-HPA)
  20. 青霉素/链霉素(100x)(PS)(Capricorn Scientific,目录号:PS-B)
  21. 淀粉状蛋白β肽1-42人(ANYGEN,目录号:AGP-8338)
  22. CM-H 2 DCFDA溶液(见食谱)
  23. MitoSOX TM Red解决方案(请参阅食谱)
  24. 聚-D-赖氨酸氢溴化物溶液(见食谱)
  25. 准备培养基(见食谱)
  26. 培养基(见食谱)
  27. 保持培养基(见食谱)

设备

  1. 血细胞计数器(MARIENFELD,目录号:0630010)
  2. 原装便携式移液助流™移液器控制器(Drummond Scientific,目录号:4-000-100)
  3. 修整剪刀(Surgimax Instruments,目录号:85-112-12)(图1A 1)
  4. 解剖剪刀(Surgimax Instruments,目录号:85-127-10)(图1A 2)
  5. 解剖剪刀(Surgimax仪器,目录号:63-175-11)(图1A 3)
  6. 春季敷料钳Sharp(Surgimax Instruments,目录号:85-076-11)(图1A 4)
  7. Spring Dressing Forceps Blunt(Surgimax Instruments,目录号:85-073-15)(图1A 5)
  8. 多用途镊子(Surgimax Instruments,目录号:05-177-11)(图1A 6)
  9. 洁净工作台(HANBAEK Scientific Technology,产品目录号:HB-402)
  10. 细胞培养CO 2培养箱(ARA,目录号:APR150)
  11. 水浴(Grant Instruments,JB Academy,产品目录号:JBA18)
  12. 离心机(Hanil Scientific,目录号:Combi 514R)
  13. 具有活细胞成像系统的共聚焦显微镜(Carl Zeiss,型号:LSM700)(图1B和1C)


    图1.实验设备A.手术器械; B.具有活细胞成像系统的共聚焦显微镜(LSM700); C.活细胞室。

软件

  1. 为了度量
    1. ZEN黑色版(蔡司共焦显微镜LSM700软件)
      注:这是ZEISS共聚焦显微镜提供的默认程序。

  2. 用于分析
    1. ZEN蓝色版(ZEISS共聚焦显微镜LSM700软件; SR-DIP软件ZEN蓝色版
    2. ImageJ( ImageJ 是一个开源的图像处理程序)

程序

在这个协议中,我们介绍了在氧化应激下原代神经元细胞中两种类型的ROS测量。

  1. 事先,用于活细胞成像的35mm板在4℃下用2ml聚-D-赖氨酸溶液涂覆24小时。
    可选:不使用35 mm培养皿进行ROS测量,可采用以下方法。将无菌玻璃盖平放在6孔板的每个孔中。向每个孔中加入2ml聚-D-赖氨酸溶液并在4℃下涂覆24小时。
  2. 取出聚-D-赖氨酸溶液并用冷PBS洗两次。

  3. 在E17大鼠胚胎正在准备时,在板凳上干燥涂层板。
  4. 使用敷料剪(设备3)和春季敷料钳Blunt(设备7)小心地从Sprague Dawley大鼠中取出E17〜E18胚胎。将它们放入装有冷的Prep培养基的培养皿中(图2A-2D)。
    注意:在这个实验中,用CO 2 2 em麻醉孕妇Sprague Dawley大鼠,并使用CO em em 安乐死>胚胎提取后的 2 。所有实验程序均经成均馆大学机构动物护理和使用委员会批准后进行。
  5. 使用解剖剪刀(设备5)和春季修整镊子夏普(设备6),仔细从子宫和胚胎袋中取出单个胚胎(图2E-2F)。
  6. 使用解剖剪刀(设备4)和多用途镊子(设备8),切除E17-E18胚胎的头皮和颅骨并拔出整个大脑。将提取的大脑置于Prep培养基中(图2G-2I)。
    注意:对于正确的细胞条件,此过程(步骤4-6)应在15-20分钟内完成。


    图2.妊娠Sprague Dawley大鼠胚胎提取过程(E17-E18)(步骤4-6)

  7. 取出胚胎的大脑后,将其置于预备培养基中,如图3A所示。
  8. 如图3B所示,解剖整个大脑的大脑皮层。
    注意:将胚胎脑部的皮层分开时,必须注意不要将其他部分分开。当您从步骤7进入步骤8(图3A至图3B)时,将微型镊子插入皮层内侧和纹状体外部之间,然后从胚胎脑切下皮层。正如您在图3A中看到的那样,皮层在包围大脑其他部分时在表面上。
    在皮质下方的内侧有纹状体。
  9. 去除大脑皮质外的脑膜和血管(图3C)。
    如图3D所示将大脑皮层置于Prep培养基中 注意:
    1. 去除如图3所示的脑膜和血管后,去除附着于皮质内侧的海马部分。您可以使用皮质部分进行皮质神经元细胞培养,或者您可以用海马部分进行海马神经元细胞培养。
    2. 对于正确的细胞条件,这个过程(步骤7-9)应该在20-25分钟内完成


    图3.从提取的全脑中提取皮层区域的过程(步骤7-9)

  10. 将大脑皮质转移到装有2ml Prep培养基的锥形管(15ml大小)中,并加入300μl胰蛋白酶溶液使最终浓度达到约10%(图4A)。
  11. 将锥形管置于37°C培养箱或水浴中,并定期轻轻敲击(图4B)。
  12. 约15分钟后,使用10毫升一次性吸管小心悬浮细胞(图4C)。

  13. 在管中加入400μlFBS至10%浓度并小心悬浮细胞。

  14. 过滤细胞通过一个70微米尼龙细胞滤网获得单细胞悬液(图4D)。
  15. 用2ml培养基稀释细胞后,在35mm培养皿中将细胞接种于1×10 6个细胞中,并将细胞在37℃培养箱中培养18小时(图4E和4F) 。

  16. 用2毫升维持培养基替换培养皿中的培养基。
    注意:如果细胞在培养基中维持时间过长,其他脑细胞类型包括小胶质细胞和星形胶质细胞很可能会长大。因此,细胞接种后12-18小时应将培养基替换为维持培养基。

  17. 将35毫米培养皿中的一半培养基换成新的1毫升维持培养基。


    图4.制造脑组织单细胞并准备细胞接种的过程(步骤10-17)


  18. 在播种后进行实验至少7天使细胞生长(图5)。
    注意:当您将细胞接种时,培养基中会出现各种脑细胞(神经元,小胶质细胞,星形胶质细胞)。然而,在用维持培养基置换后,只有神经元细胞保持特异性,因为除了神经元细胞以外的所有其他类型的细胞都被构成维持培养基的B-27除去。


    图5.第1天(A)和第8天(B)后的细胞形态。比例尺,A =500μm; B = 200微米。


  19. 在ROS诱导条件下孵育细胞约24小时 注意:在本方案中添加5μM低聚Aβ1-42 em / em是为了诱导ROS,但它可能因您的实验条件。 Aβ1-42已知可诱导ROS(Andrey等人,2004,Shelat等人,2008)。 >
  20. 然后用1μlCM-H 2 DCFDA溶液(5mM)或MitoSOX TM Red溶液(5mM)处理20分钟。
    注意:MitoSOX TM 是线粒体超氧化物和CM-H 2 2 than superoxide(O 2 - < / SUP> )。因此,即使两种化学品同时加工,它们也会被标记不同的ROS。然而,难以区分两种类型的ROS并分析共焦图像中的荧光。我们建议分开准备样品并进行每个实验。
    可选:本部分提供了另一种准备样本以捕获活细胞图像的选项。如果您在盖玻片上培养细胞,请仔细将盖玻片放入新的35毫米活细胞成像皿中。将细胞贴在底面的盖玻片侧面
  21. 用新的2毫升维持培养基更换。
    注意:预先设定活细胞成像的条件; 37°C和5%CO 2 是必需的,因为我们希望保持原代神经元细胞的生长条件。
  22. 使用共焦显微镜,测量由MitoSOX TM(510 / 580nm,参见图6)或CM-H 2 DCFDA(495 / 520nm,参见图7 )。由于荧光不会持续很长时间,因此建议在30分钟内测量。


    图6.用MitoSOX TM Red检测大鼠原代神经细胞线粒体中的超氧化物用超氧化物氧化MitoSOX TM Red试剂产生红色荧光。 A.明场大鼠原代细胞图像; B.超氧化物产生的红色荧光; C.合并图像。比例尺= 50微米。


    图7.使用CM-H 2 DCFDA测量在大鼠原代细胞中由Aβ诱导的细胞内区室中的ROS。绿色荧光表示Aβ或媒介物处理的样品的细胞内ROS水平。 A.载体处理的样品; B.5μM的Aβ处理的样品。比例尺= 50微米。

    共聚焦图像测量设置
    您可以按照图8中的流程使用共聚焦显微镜测量ROS的水平:
    1. 运行Zen黑版。
    2. 点击'Acquisition'选项卡中的'Smart Setup'按钮,选择合适的激发/发射波长(MitoSOX TM或CM-H 2 DCFDA)(图8 A1)。
    3. 设置染料的适当激发/发射波长,并在“搜索”选项卡中选择图像颜色(图8C 2)。
    4. 点击“最佳信号”按钮。
    5. 将样品放在活细胞成像室(图1C)。
    6. 点击'定位'标签并调整共聚焦显微镜的焦点(图8B 3)。
    7. 设置显微镜的焦点后,点击'采集'标签中的'设置曝光'按钮。
      注意:'设置曝光'会自动调整检测器的增益值(图8A)。
    8. 点击“Live”按钮(图8A)。
      注意:'Live'会持续扫描实时图像。
    9. 要在'实时'条件下获得更清晰和更准确的图像,请调整'增益','数字偏移','数字增益','针孔'和激光功率(图8D 5)的每个值。您还可以调整“速度”和“平均”以获得更好的图像。
      注意:在这个实验中,“速度”设置为7,“平均”数量设置为8.(图9)
    10. 点击'Snap'按钮获取图像(图8A)。
      注:所有样品应在相同条件下测量。测量方法基于共焦设备的说明。


    图8. ZEN黑色版图像测量程序的测量方法流程图

    “”src
    图9.用于在ZEN黑色版本中控制“速度”和“平均”的选项卡

数据分析

测量ROS的共焦图像可以通过量化荧光强度用于统计分析。该协议提供了两种方法。

  1. 使用基本共焦驱动程序的测量方法(见图10)。


    图10. ZEN蓝色版本的分析流程图运行由Zeiss共焦设备提供的分析工具Zen Blue版本。打开要分析的图像,然后单击“分析”选项卡。按'Setup Image Analysis'按钮设置分析方法(见图10-1)。按顺序输入正确的分析条件。特别是,设置适当的阈值(当您点击CM-H <2> DCFDA发出荧光的区域时,将自动设置默认值)(必须设置分析设置值基于阳性对照设置,所有待分析图像的设置应同样适用)。确定要测量的区域,如图10-4所示。设置想要获得的结果(荧光平均值,标准偏差,荧光点数,等等),然后按'完成'按钮。最后,按'分析'按钮检查值。

  2. 使用ImageJ的测量方法(见图11)


    图11. ImageJ的分析流程图要使用ImageJ测量荧光强度,需要执行几个预备步骤。打开要分析的图像后,必须分离要测量的荧光区域(在流程图3中,在调整阈值时设置要测量的范围,参见图11-2,11-3)。点击如图4所示的“设置测量”选项卡,设置您想要获得的结果。点击“测量”标签并获得结果。

笔记

  1. 在测量荧光之前20分钟,应用MitoSOX TM或CM-H 2 DCFDA处理每个实验组。您不应该在实验样品中同时处理MitoSOX TM和CM-H 2 DCFDA(步骤19)。
  2. 如果不维持细胞生长条件,则ROS测量值将不准确。所以,在拍摄共焦图像之前,至少要等待10分钟的稳定时间(步骤21)。
  3. 当培养原代神经元时,在细胞接种后延迟培养基更换导致培养细胞中神经元百分比的降低(步骤16)。

食谱

  1. CM-H 2 DCFDA溶液(5mM)

    在17μlDMSO中溶解50μgCM-H 2 DCFDA(50μg/ 1瓶)
  2. MitoSOX TM Red溶液(5 mM)

    在13μlDMSO中溶解50μgMitoSOX TM Red(50μg/ 1瓶)
  3. 聚-D-赖氨酸氢溴酸盐溶液
    聚-D-赖氨酸氢溴酸盐(5毫克/瓶)
    50毫升无菌超纯水
  4. 准备培养基
    PBS 45毫升
    5毫升PS
  5. 培养基
    500毫升DMEM
    50毫升FBS
    5毫升PS
  6. 维护培养基
    50毫升Neurobasal媒体
    1毫升B-27补充剂
    500μlPS

致谢

这项研究得到了基础科学研究计划通过韩国国家研究基金会(NRF)资助的赠款(2012R1A5A2A28671860,2017M3C7A1048268)的支持,韩国教育,科学和技术部。作者声明他们没有利益冲突要申报。

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Baek, S. H., Cho, Y., Lee, J., Choi, B. Y., Choi, Y., Park, J. S., Kim, H., Sul, J., Kim, E., Park, J. H. and Jo, D. (2018). Intracellular and Mitochondrial Reactive Oxygen Species Measurement in Primary Cultured Neurons. Bio-protocol 8(11): e2871. DOI: 10.21769/BioProtoc.2871.
  2. Baek, S. H., Park, S. J., Jeong, J. I., Kim, S. H., Han, J., Kyung, J. W., Baik, S. H., Choi, Y., Choi, B. Y., Park, J. S., Bahn, G., Shin, J. H., Jo, D. S., Lee, J. Y., Jang, C. G., Arumugam, T. V., Kim, J., Han, J. W., Koh, J. Y., Cho, D. H. and Jo, D. G. (2017). Inhibition of Drp1 ameliorates synaptic depression, Aβ deposition, and cognitive impairment in an Alzheimer's disease model. J Neurosci 37(20): 5099-5110.
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